Project Summary Bacteria that chronically colonize the host, such as the gut microbiota, must adapt to various forms of stress in the host environment. The molecular mechanisms bacteria use to sense and respond to these environmental signals are crucial for maintaining symbiotic associations with host cells. Oxidative stress is a hallmark of host- microbe interaction best known for its role in the host immune response; however, epithelial barriers also generate reactive oxygen species (ROS) in response to microbial contact. My lab uses chemical and genetic tools to define molecular mechanisms of bacterial adaptation to oxidative stress. We use the common gastric symbiont Helicobacter pylori to model bacterial responses to physiological ROS. H. pylori is a normal member of the gastric flora that can persist for decades in the host despite constant exposure to ROS-generating epithelial cells, similar to many commensal microbes. Using H. pylori co-cultured with gastric epithelial cells, we have developed a chemical proteomic strategy that can identify protein targets of ROS at the host-microbe interface. Unlike conventional methods for analyzing changes in gene expression, our approach detects post- translational oxidative modifications that can alter cell signaling even when protein levels do not change. This allows us to uncover redox-signaling events at the host-microbe interface that are largely unexplored and likely mediate bacterial adaptation to oxidative stress. In parallel, we are investigating thiol-containing small molecules that maintain redox balance within bacterial cells. While these low-molecular-weight (LMW) thiols are synthesized by nearly all life forms, certain classes of bacteria lack the canonical enzymes required for LMW-thiol biosynthesis. Consequently, how these bacteria detoxify ROS at the host-microbe interface remains an open question. We recently discovered a novel bacterial transporter of ergothioneine (EGT), an LMW thiol with potent antioxidant properties that is abundant in animal tissues. This transporter is broadly conserved in bacteria that commonly colonize the gastrointestinal tract; thus, EGT uptake could represent a new mechanism of microbial redox regulation at the host-microbe interface. In this proposal, we will determine how protein oxidation and LMW-thiol transport shape bacterial adaptation to the host environment. First, we will identify bacterial proteins that are oxidized following microbial contact with ROS-generating eukaryotic cells and elucidate the redox-signaling pathways that enable bacterial adaptation to physiological ROS. Second, we will characterize the proteins responsible for EGT transport in bacteria to increase understanding of this highly conserved process and its role in microbial redox biology. Together, these studies will define fundamental redox-signaling pathways (project 1) and transport mechanisms (project 2) that help maintain homeostasis at the host-microbe interface. In the long term, our work will provide a framework for investigating these processes in other microbes and could reveal new targets for the rational design of anti-infective therapies.